Ubiquitination mediated by E1-E2-E3 multi-enzyme cascades rivals phosphorylation as a predominant mechanism regulating myriad protein functions (Cohen and Tcherpakov, 2010; Nalepa et al., 2006). Repeated catalytic cycles result in substrates modified on multiple lysines with various polyubiquitin chains, which alter protein functions in an extraordinary variety of ways. Because E3 ligases control substrate specificity and the topology of ubiquitination, they represent attractive targets for therapeutic intervention (Nalepa et al., 2006; Petroski, 2008). Yet, identifying the diversity of mechanisms regulating E3 ligases, as well as generation of tools for their manipulation, has lagged behind deciphering regulation and developing therapeutics for kinases (Cohen and Tcherpakov, 2010; Nalepa et al., 2006). The first family of E3 ligases discovered (Huibregtse et al., 1995), HECT (Homologous to E6AP C-Terminus) E3s, have been directly implicated in cancer, hypertension, neurological disorders, and other diseases (see Table 2, below) (Rotin and Kumar, 2009; Scheffner and Kumar, 2014). Moreover, some pathogenic bacteria have evolved HECT-like E3s as virulence factors to manipulate host cell signaling (Lin et al., 2012; Rohde et al., 2007). Therefore, understanding molecular mechanisms of HECT E3 function could greatly advance therapeutic strategies for many diseases.
Development of agents to selectively modulate HECT E3s has been hampered by extreme inter-domain rotations accompanying catalysis, a shallow active site, and dynamic regulation of HECT E3 activity (Escobedo et al., 2014; Gallagher et al., 2006; Huang et al., 1999; Kamadurai et al., 2013; Kamadurai et al., 2009; Mari et al., 2014; Persaud et al., 2014; Ronchi et al., 2013; Verdecia et al., 2003; Wiesner et al., 2007). In principle, recently reported small molecule and peptide inhibitors obtained by high throughput screening for several HECT E3s provide routes to assess functions and mechanisms of HECT E3s in normal and diseased cells (Cao et al., 2014; Kathman et al., 2015; Mund et al., 2014; Rossi et al., 2014). However, existing molecules generally do not conform to a general strategy that could be used to interrogate HECT E3s across the family, fall short in terms of potency and specificity, and generally have had limited utility in probing unknown HECT mechanisms.
The defining feature of HECT E3s is a ˜40 kDa C-terminal “HECT domain” containing two flexibly-tethered lobes (N- and C-), with 16-92% amino acid identity across the family. In addition to the catalytic domain, HECT E3 primary sequences reveal various N-terminal domains that may enable substrate binding and dynamic regulation by mediating autoinhibition and influencing subcellular localization (FIG. 1A). The largest and best-characterized class of HECT E3s comprises the NEDD4-family, which display a common architecture consisting of an N-terminal C2 domain, 2-4 central WW-domains distal and proximal to the catalytic domain, and the C-terminal HECT domain (Rotin and Kumar, 2009; Scheffner and Kumar, 2014) (FIG. 1A).
Studies of E3s in the NEDD4-family revealed that the HECT domain interacts with Ub at multiple sites. For example, in complex with E2˜Ub or in the E3˜Ub intermediate, the HECT “C-lobe” binds the Ub to be transferred, and a separate C-lobe interaction with the acceptor Ub is implied from biochemical studies (Kamadurai et al., 2013; Kamadurai et al., 2009; Kim and Huibregtse, 2009; Maspero et al., 2013). In addition to interactions made by the active-site-bound Ub, a weak Ub-binding “exosite” has been reported in the HECT “N-lobe” of various NEDD4-family E3s (French et al., 2009; Kim et al., 2011; Maspero et al., 2011; Ogunjimi et al., 2010).